Amino Acid 261 in the alpha subunit of RNA polymerase is the most critical for CAP-dependent transcription in vivo. To confirm and quantify the in vivo experiments we subcloned the genes encoding [Gly 261] a and [Lys 261] a into T7 RNA polymerase expression vector pET3a, purified the mutant a subunits and wild type b, b1 and subunits and reconstituted RNA polymerase. We have determined the purity of mutant a subunit by mass spectrometry. A paper describing this work has been published in Gene Dev..